pH-gated nanoparticles selectively regulate lysosomal function of tumour-associated macrophages for cancer immunotherapy

Tumour-associated macrophages (TAMs), as one of the most abundant tumour-infiltrating immune cells, play a pivotal role in tumour antigen clearance and immune suppression. M2-like TAMs present a heightened lysosomal acidity and protease activity, limiting an effective antigen cross-presentation. How to selectively reprogram M2-like TAMs to reinvigorate anti-tumour immune responses is challenging. Here, we report a pH-gated nanoadjuvant (PGN) that selectively targets the lysosomes of M2-like TAMs in tumours rather than the corresponding organelles from macrophages in healthy tissues. Enabled by the PGN nanotechnology, M2-like TAMs are specifically switched to a M1-like phenotype with attenuated lysosomal acidity and cathepsin activity for improved antigen cross-presentation, thus eliciting adaptive immune response and sustained tumour regression in tumour-bearing female mice. Our findings provide insights into how to specifically regulate lysosomal function of TAMs for efficient cancer immunotherapy.


REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): with expertise in nanomedicine, cancer immunology In the manuscript authors developed a pH-gated nanoparticle (PGN) adjuvant that selectively targets the lysosomes of M2-like TAMs in tumours.The authors show convincing data that they were able to design a pH-gated nanoparticle, PGN4.9, that only opens in M2 macrophages both in vitro and in vivo.In addition, using PGN4.9-deliveredimidazoquinoline (IMDQ), they were able to differentiate M2 into M1 macrophages in the tumor microenvironment.PGN4.9 IMDQ itself lead to a reduced tumor growth but could also be combined with other immune therapies to further decrease tumor growth.
The findings are novel, original and well-supported by the data.

Specific comments:
One issue that should be addressed is that the text is written in an overcomplicated manner which makes it difficult to understand some sections (example: lines 84-96).Secondly, in this manuscript the authors designed a delivery method for an immunomodulatory drug.
The term "nanoparticle adjuvant" or "PGN adjuvant" frequently used in the manuscript is misleading, since the adjuvant is the nanoparticle drug cargo and not the particle itself.
Figure 2, please show that the particles are taken up specifically in the lysosome (for instance by costaining).2D) Please choose cells with the same amount of particles (Cy3.5)inside the cell.3D) please discuss why PGN4.3 and PGN4.9 accumulate but only PGN4.9 and PGN6.3 activate.3E) Please discuss why the particles don't open in the other organs harboring M2 macrophages (liver/spleen).In this figure no controls, or other particles, are shown.Please also show this data for the other particles.3F) For clarity, please use the same colors between pie chart and confocal pictures.3J) the word 'dramatic' is exaggerated 4B) please call it a reporter assay in the text rather than RAW blue.In my understanding, for the functionality of the particle, the lysosomal pH is very important.If so, please show the pH of the RAW lysosome.SI21)a, m1 and m2 are the untreated cells?And the particles are tested on which type of macrophages?please clarify.SI22) Please explain why the H&E staining shows good bio compatibility 5A/B/C) Please also show the raw count rather than only percentages SI32D) Please discuss why there are equal amounts of M2 macrophages between the IMDQ, NPGN and PGN4.9.5G) Please explain how the CFSE dye correlates with killing.6I/J) As the PDPA-DTX is not an established treatment that increases antigen presentation, please show this.It would also be interesting to see immune infiltration data for this experiment to determine how it worked.Literature describes that activating existing M1 macrophages using imidazoquinoline TLR ligands can inhibit tumor growth.The authors should discuss why only targeting M2 macrophages would be more beneficial rather then targeting both type of macrophages.
The work has not been put into perspective in the final section of the manuscript.It is important that the authors show the relevance in comparison to current literature on this topic.

Reviewer #2 (Remarks to the Author): with expertise in tumor associated macrophages
Tang et al present pH-gated nanoparticles, termed PGN4.9, that degrade in the lysosomal compartment of M2-oriented macrophages, but not M0/M1 or other normal cell types or cancer cells.The particles accumulate in the tumor microenvironment and, when loaded with the TLR7/8 ligand IMDQ, are able to skew the macrophage activation state to M1, with a higher antigen-presenting capacity and anti-tumor activities.Consequently, PGN4.9 collaborates with anti-PD1 to increase anti-tumor immunity.His is an interesting story, but some remarks remain: 1) I don't understand why PGN6.3 would accumulate less in the tumor.Accumulation is influenced by size, stability etc, so is this different for PGN6.3?
2) The authors conclude that PGN4.9 does not get activated in normal tissues, based on observations in the liver and the spleen.However, the authors should look at organs where more M2-oriented macrophages are likely to reside, such as eg the lung and possibly also the skin 3) Fig 3g shows a correlation between the ratiometric signal and the presence of CD206+ TAMs.However, tumors may also contain M1-like TAMs.How is the correlation with those TAMs?The authors should more directly demonstrate that PGN4.9 fluoresces only within the more M2-oriented TAMs 4) Fig 4b also shows that PGN4.9 adjuvant (I presume that this is the PGN4.9 particle conjugated to IMDQ, although I don't find this very clear from the text) stimulates TLR a lot less than free IMDQ.Please explain.
5) The experimental conditions used in Figure 4 should be clearer represented on the figures or in the legend.If the authors use "PGN4.9",what does that mean?I presume they mean M2 + PGN4.9, but that's nowhere stated.Could also be M0 + PGN4.9.

Reviewer #3 (Remarks to the Author): with expertise in nanoparticles
In this manuscript, a pH-gated nanoparticles library was established for selectively target the highly lysosomal acidity of M2-like macrophages.The PGN4.9 nanoadjuvant with ANDgate capacity can selectively fine-tune lysosomal proteolysis of M2-like TAMs to facilitate antigen cross-presentation and provoke adaptive tumour immunity.The study is interesting, but some results are not very persuasive.Based on these points, I suggest that this manuscript can be accepted for publication after addressing following issues.
1. Line 437, the authors adopted fluorescence intensity to evaluate the PGN accumulation and activation efficiency.This method of quantitative analysis is not accurate.The PNG with the fluorescent label attached may have residual fluorescent label, or the fluorescent label may be free from the polymer.To assess the accumulation or metabolism of PNG, a more accurate method is still chromatography or chromatography-tandem mass spectrometry.
2. After PGN4.9 was injected into animals through the tail vein, how does it target the M2like TAM in tumors?What is the mechanism of this targeting function?How was it considered when designing the synthesis?3.By using what kind of method to verify the lysosomal pH value in BMDMs to obtain the fitting standard curve?Can you provide more experiment protocols?4. The manuscript contains too many abbreviations, which makes the reading process not very smooth.The meaning of the acronym is often forgotten.If the phrase or word composition is not complex, try to use the original expression to make the article reading more smoothly.If a phrase occurs only once or twice, it is not necessary to assign a separate abbreviation.For example, TME stands for tumor microenvironment, APCs for antigenpresenting cells, and GPC for chromatography.It is not necessary to create an abbreviation for these phrases or words.5.In supplementary Fig. 5, in the plot, the author should elaborate what is the F instead for.
End explain the Two coordinate system in accuracy, avoiding ambiguous understanding.
What F/Fmin instead for?6.In supplementary Fig. 5, the fluorescence intensity of Cy3.5 is stable, while the BDP is fluctuate dramatically.Why? 7. In Line 422, "tumors were frozen and cut into several adjacent slices with 10 μm thickness".Do you mean "Tumor tissues were cryo-sectioned and sliced into 10-micron slices."The type of instrument that used, and the freezing temperature should be described.9 -In line 254, the sentence "The effects of PGN4.9 on tumour inhibition was mitigated obviously in macrophages-depleted mice" must be reformulated, because the "mitigation" is not so obvious indicating that other cell types are also important..It could be also mentioned that macrophage depletion is not complete with the CSF1R antibody.10 -For supp.fig.38 and fig.6i, the timing and doses of each therapeutic approach are not clear.These should be indicated.
11 -Discussion must be significantly improved according with the following comments.a)In the first paragraph of discussion more references are needed.b) More details and references about the nanotechnology would be appreciated (other pH sensitive NPs).c) drug delivery approach to target macrophages in tumors require appropriate discussion.

Point-by-point response to reviewers
We would like to thank the reviewers for the insightful and constructive comments!We have revised the manuscript according to their advices, which should significantly improve the clarity and quality of our work.Below is a list of the point-by-point responses to the reviewer comments shown in italics and the corresponding changes that we made highlighted in yellow.

Reviewer #1 (Remarks to the Author): with expertise in nanomedicine, cancer immunology
In the manuscript authors developed a pH-gated nanoparticle (PGN) adjuvant that selectively targets the lysosomes of M2-like TAMs in tumours.The authors show convincing data that they were able to design a pH-gated nanoparticle, PGN4.9, that only opens in M2 macrophages both in vitro and in vivo.In addition, using PGN4.9-deliveredimidazoquinoline (IMDQ), they were able to differentiate M2 into M1 macrophages in the tumor microenvironment.PGN4.9 IMDQ itself lead to a reduced tumor growth but could also be combined with other immune therapies to further decrease tumor growth.The findings are novel, original and well-supported by the data.

Specific comments:
One issue that should be addressed is that the text is written in an overcomplicated manner which makes it difficult to understand some sections (example: lines 84-96).Secondly, in this manuscript the authors designed a delivery method for an immunomodulatory drug.The term "nanoparticle adjuvant" or "PGN adjuvant" frequently used in the manuscript is misleading, since the adjuvant is the nanoparticle drug cargo and not the particle itself.
We appreciate the Reviewer's encouraging comments on the novelty and impact of the PGN nanotechnology.We have revised the manuscript according to your advices and other referee's comments.
We have rewritten the mentioned sections to help understand the nanoparticle characterization experiment.Besides, we have corrected the original expression and made some changes in the revised manuscript.These changes will not influence the content and framework of the paper.
Lines 85-89: Furthermore, the pHt of PGNs was determined to be within a range from 4.46 to 5.47 with sharp response (∆pHON/OFF ~ 0.2-0.3,Fig. 2b) using fluorescence analysis procedure 21 .To evaluate the pH-gating capacity of PGNs in living cells, we successfully constructed binary ratiometric nanoreporters, which consisted of the 'OFF-ON' and 'Always-ON' fluorescence modules in visible-light window 24 .
1. Figure 2, please show that the particles are taken up specifically in the lysosome (for instance by co-staining).2D) Please choose cells with the same amount of particles (Cy3.5)inside the cell.
Re: Per the reviewer's advice, we have conducted the intracellular trafficking experiments on BMDMs co-staining with Lysotracker red, which is an indicator of lysosome.As shown in Supplementary Fig. 7, the yellow signals indicated the colocalization of PGN4.9 with lysosomes in BMDMs with different phenotypes.
We added the above results in Supplementary methods and Supplementary Figure 7. Lines 99-101: We firstly imaged the intracellular trafficking of PGNs after incubation with living cells for 2 h.Results showed that PGNs had a good colocalization with lysosomes (Supplementary Fig. 7).
Per the reviewer's suggestion, we have replaced the original images with new representative confocal images for M1-and M2-like macrophages in Figure 2d.As for the M0-like macrophages, the lysosome number is pretty low due to its poor phagocytic activity.Thus, only small amount of Cy3.5-positive organelles were captured.

Fig 3, Please also show lysosomal uptake in vivo.
Re: We acknowledge the reviewer's kind suggestion.In order to investigate the distribution of nanoparticles in vivo, we performed immunofluorescence staining of Lamp 1 in tumour tissue sections to image the lysosome trafficking of PGN4.9.As shown in Supplementary Fig. 13, we observed that a majority of nanoprobes were trapped in lysosomes at 24 h post-administration, as indicated by the good colocalization with Lamp 1.
We added the above results in Supplementary methods and Supplementary Figure 13.Lines 124-128: To evaluate the lysosomal accumulation of PGNs in vivo, 4T1-tumour bearing mice were intravenously injected with Cy5-conjugated PGN4.9, and tumours were excised at 24 h post-injection and cryosectioned for the immunofluorescence staining of LAMP 1 (lysosome biomarker) in tumour slices.
Results demonstrated that PGNs had a good colocalization with lysosomes within tumour tissues at 24 h post-administration (Supplementary Fig. 13).
Re: According to our previous studies (Li et al, Nanomedicine 2019, 17: 287-296), All three ICG-conjugated micelles had long blood circulation with half-lives of more than 10 h, while the pHt of nanoparticles have significant effect on their blood AUC0-24 h.Moreover, our group performed an in-depth research on the effect of hydrophobicity of micellar core on its pharmacokinetics behavior, finding that lower pHt of nanoparticles would lead to longer circulation time in vivo, as shown in Figure R1.Therefore, the tumour accumulation in PGN4.9-andPGN4.3-treated groups was 1.93-and 2.06-fold of that in PGN6.3-treated group due to the higher blood AUC for PGNs with lower pHt.On the other hand, we determined that the lysosomal pH of different cell types within tumour microenvironment is higher than 4.4 (pHL > 4.4).Thus, PGN4.3 is hard to respond to lysosomal pH of M2-like TAMs, resulting in a dim fluorescence signal, whereas PGN6.3 and PGN4.9 can respond to the acidic signals within lysosomes and caused complete signal activation for lighting up tumour.Take these two factors into account, PGN4.9 can achieve both higher tumour accumulation and intracellular activation as compared with the other two PGNs.

3E) Please discuss why the particles don't open in the other organs harboring M2 macrophages
(liver/spleen).In this figure no controls, or other particles, are shown.Please also show this data for the other particles.

Re:
Our previous work has demonstrated that although the nanoparticles accumulated a lot in mononuclear phagocyte system (e.g., liver and spleen) over 24 h post-administration, the internalized amounts of nanoparticles only account for a very low percentage (3-5%) due to its limited endocytosis efficiency by cells (macrophage and hepatocytes) within liver and spleen.In contrast, about 20% of tumour accumulated nanoparticles were internalized by cells within tumour tissues (Yin et al, Nat Commun 2021, 12, 2385).As shown in Figure R2, the most internalized NPs were not colocalized with macrophages in liver, which suggested that nanoparticles cannot efficiently endocytosed by macrophages in liver and spleen, unlike the behavior in tumour tissues.
Per the reviewer's advice, we have carried out new experiments about the accumulation level and activation efficiency in PGN6.3 and PGN4.3 control groups.The readouts of PGN6.3 and PGN4.3 activation efficiency in livers and spleens with macrophages were still significantly lower than that in tumour tissues.
We added the above results in Supplementary methods and Supplementary Figure 17.

3F) For clarity, please use the same colors between pie chart and confocal pictures.
Re: Per the reviewer's suggestion, we have revised the color coding across panels in Figure 3f in the revised manuscript.

3J) the word 'dramatic' is exaggerated
Re: Thanks for the suggestion.We have revised this sentence according to the statistical analysis.Line 158-160: Moreover, depletion of 58.8% M2-like TAMs with clophosome caused a significantly decreased signal activation of PGN4.9, further elucidated the selective targeting of PGN4.9 to M2-like TAMs (Fig. 3h-j; Supplementary Fig. 20).7. 4B) please call it a reporter assay in the text rather than RAW blue.In my understanding, for the functionality of the particle, the lysosomal pH is very important.If so, please show the pH of the RAW lysosome.

Re:
We appreciate the reviewer's advice and have changed the manuscript on RAW blue.We have measured the pH value of RAW-Blue with or without cytokine IL-4 by the ratiometric pH quantification, as shown in Figure R3.The results showed that the lysosomes of RAW-Blue with IL-4 (pHL ~ 4.50) were hyper-acidified as compared with untreated RAW-Blue (pHL ~ 5.21).Re: Thanks for the reviewer's good suggestion.To avoid misleading, we have revised the legend in Supplementary Figure 25.In the reprogramming experiments, we incubated different groups of immune adjuvants with M2-like BMDMs, while M1-and M2-like BMDMs were the untreated cells and chosen as control groups.

SI22) Please explain why the H&E staining shows good bio compatibility
Re: Thanks for the reviewer's question.Compared with PBS group, major organs from mice treated with different formulations kept tissue structure intact without cell morphology change.Moreover, no necrosis and fibrosis were observed in the major organs.Thus, the H&E staining shows good biocompatibility of different nanoadjuvants.We have added the explanation in the main text.
Line 182-184: Hematoxylin and eosin (H&E) staining of major organs showed that pretreated mice kept tissue structure intact without cell morphology change in major organs, indicating a good biocompatibility of PGN4.9 nanoadjuvant (Supplementary Fig. 26).

5A/B/C) Please also show the raw count rather than only percentages
Re: Thanks for your kind advice.We have added the raw count of immune cells to compare the lymphocyte infiltration in tumours between different treatments as shown in Supplementary Figure 37.  11.SI32D) Please discuss why there are equal amounts of M2 macrophages between the IMDQ, NPGN and PGN4.9.
Re: Re: The reviewer raised a good point.Because of the high efficiency of TLR activation by IMDQ with a very low EC50 (Rodell et al, Nat Biomed Eng 2018, 2: 578-88), all the three IMDQ preparations down-regulated the M2-like tumour-associated macrophages.
However, unlike PBS, IMDQ and NPGN nanoadjuvant control groups, PGN4.9 nanoadjuvant could sustainably release large numbers of IMDQ drug for 48 h in vivo.Thus, PGN4.9-treated mice presented a dramatically higher intratumoural ratios of M1-like TAMs to M2 subset, to which we paid more attention according to the reported literatures (Wang et al, Sci Tran Med 2021, 13(615): eabb6981).As for the equal level of M2-like macrophage in different groups, it is probably due the feedback regulation after the macrophage repolarization (Chen et al, Sig Transduct Target Ther 2023, 8, 207).

5G) Please explain how the CFSE dye correlates with killing.
Re: Thanks for the reviewer's question.The carboxyfluorescein succinimidyl ester (CFSE) method has been extensively utilized to quantify the OVA-specific CTL response (Luo et al, Nat Nanotechnol 2017, 12,  648-654).Ovalbumin (OVA) was used as a model antigen.On the third day after the last treatment, immunized mice were intravenously injected with an equal number of OVA257-264-presented splenocytes (CFSE high ) and naive splenocytes (CFSE low ).Mice with strong immunity can recognize OVA-presented splenocytes labelled with high level of CFSE (CFSE high ) for specific killing, while the naive splenocytes (CFSE low ) cannot be killed by OVA-specific CTL.Therefore, we investigated the changes in the proportion of two groups of splenocytes with high and low expression of CSFE to calculate the OVA-specific killing capacity.

6I/J) As the PDPA-DTX is not an established treatment that increases antigen presentation, please show
this.It would also be interesting to see immune infiltration data for this experiment to determine how it worked.Re: Thanks for the reviewer's question.The chemotherapeutic PDPA-DTX nanomedicine can directly kill tumour cells, triggering immunogenic cell death and tumour-associated antigen release, which has been proved in our previous studies (Du et al, Adv Funct Mater 2020, 30(39): 2003757).Thus, the nanomedicine can in situ supply abundant tumour antigen and recruit macrophages to promote antigen cross-presentation in tumour tissues.When combined with PDPA-DTX nanomedicine, PGN4.9 further boosted intratumoural CD3 + CD8 + T cells.
We also performed the new experiments to determine the immune cell infiltration in tumours treated with PDPA-DTX, or PDPA-DTX plus PGN4.9 nanoadjuvant.The immune infiltration data for the combination therapy have been included in Supplementary methods and Supplementary Figure 44.

6K) Why the sudden switch from 4T1 to MC38 in this figure? All previous experiments were only demonstrated for 4T1. Did they also work with MC38?
Re: Thanks for the reviewer's question.We have already confirmed the pH-gated imaging of intratumoural M2-like macrophages in MC38 tumour model as shown in Figure 3g and Supplementary Figure 19.In addition, we performed antigen presentation experiments in lymph node and specific T cell killing experiments from splenocytes in MC38 tumour model as shown in Figure 5e-5j.To investigate the immunological memory effect, the MC38 tumour model was selected since 6 out of 10 MC38 tumours were completely removed after PGN4.9 nanoadjuvant treatment, allowing for long-term rechallenge experiments.the combination therapy.For the PGN4.9 group, 2.90-fold higher percentage of CD3 + CD8 + leukocytes were observed as compared to the PBS group.The combination with anti-PD1 antibody further promoted the intratumoural infiltration of CD8 + T cells, which was 4.87-fold more than that in PBS treated tumours, with negligible influence on CD4 + CD25 + regulatory T cells.We included the infiltrating leukocytes studies in Supplementary methods and Supplementary Figure 46.
Line 291-294: The results showed that α-PD1 treatment alone led to no significant inhibition of tumour growth, whereas 70% of the mice immunized with α-PD1 and PGN4.9 nanoadjuvant exhibited no tumour progression within 70 days by significantly promoting the intratumoural infiltration of CD8 + T cells (Fig. 6l-6n; Supplementary Fig. 46).16.Literature describes that activating existing M1 macrophages using imidazoquinoline TLR ligands can inhibit tumor growth.The authors should discuss why only targeting M2 macrophages would be more beneficial rather than targeting both type of macrophages.
Re: Thanks for the reviewer's good question.We agree that targeting M1-and M2-like TAMs is more beneficial for tumour inhibition.However, it is hard to specifically activate M1-like TAMs rather than M0and M1-like macrophages in normal tissues.M2-like phenotype accounts for 60%-90% of tumour-associated macrophages in different tumour models.Targeting M2-like TAMs using PGN4.9 nanoadjuvant can not only repolarize M2-like phenotype to M1 ones, but also trigger the sustained release of IMDQ for persistent activation of the function of reprogrammed M1-like macrophages.Meanwhile, the released IMDQ probably plays a bystander effect for the activation of M1-like TAMs.Thus, PGN4.9 nanoadjuvant achieves targeting both type of macrophages for tumour inhibition.Combining the next question (Q17), we have added the discussion in the main text.17.The work has not been put into perspective in the final section of the manuscript.It is important that the authors show the relevance in comparison to current literature on this topic.
Re: Thanks for the reviewer's suggestion.We have discussed the relevance in comparison to published literatures in the revised manuscript.
Line 313-336: Immunoagonists (e.g.R848 and CpG) in free form are easily distributed throughout the body and cause severe systemic immunotoxicity, which hampers their clinical translation 39,40 .A variety of nanoparticles have been developed to deliver immunoagonists and achieve the polarization of M2-like TAMs 10,41,42 .Recent studies demonstrated that intravenous delivery might increase the possibility of effective co-localization of immunologic adjuvant with dying tumour cells, thus producing an in situ vaccination for superior immune responses 43 .However, many such strategies based on the tissue targeting mechanism that could also activate M0-and M1-like macrophages in non-malignant organs including liver, spleen, lung and skins, raising biosafety concerns.Therefore, nanoadjuvant technology that specifically modulate M2-like TAMs would provide safe and effective formulations optimized for cancer immunotherapy.
Based on our finding that the lysosomal pH difference between M2-like TAMs (pHL ~ 4.4) and M0and M1-like macrophages (pHL ~ 5.2), engineering of pH-responsive nanotechnology would be a promising approach to achieve targeted modulation of M2-like TAMs.So far, pH-responsive nanoparticles have been extensively reported to shuttle the therapeutic cargoes to solid tumours through pH-triggered nanocarrier disintegration or linkage cleavage upon pH changes within tumour microenvironment [44][45][46] .Although these approaches offer the targeted delivery of therapeutic cargoes to lysosomal compartments, achieving selective targeting to the highly lysosomal acidity of M2-like TAMs rather than that of other cells remains a significant challenge.In this article, our PGN nanoadjuvants were successfully designed and screened with several key features that enable the specific polarization of M2-like TAMs instead of other macrophages in normal tissues due to their AND-gated performance.Firstly, the PGN nanoadjuvants render a sharp pH response (∆pHON/OFF ~ 0.2-0.3),which is critical for the pH-gated activation in lysosomal compartment of M2-like TAMs rather than counterpart of other macrophages.Secondly, the pHt tunability enables the successful screening of PGN4.9 nanoadjuvant for the specific targeting of lysosomal pH of M2-like TAMs, followed by enzymatic cleavage-mediated drug release to achieve logic-gated immunotherapies.
Reviewer #2 (Remarks to the Author): with expertise in tumor associated macrophages Tang et al present pH-gated nanoparticles, termed PGN4.9, that degrade in the lysosomal compartment of M2-oriented macrophages, but not M0/M1 or other normal cell types or cancer cells.The particles accumulate in the tumor microenvironment and, when loaded with the TLR7/8 ligand IMDQ, are able to skew the macrophage activation state to M1, with a higher antigen-presenting capacity and anti-tumor activities.Consequently, PGN4.9 collaborates with anti-PD1 to increase anti-tumor immunity.His is an interesting story, but some remarks remain: We thank the reviewer's positive comments and provide a point-by-point response to the reviewer comments.
1.I don't understand why PGN6.3 would accumulate less in the tumor.Accumulation is influenced by size, stability etc, so is this different for PGN6.3?
Re: According to our previous studies (Li et al, Nanomedicine 2019, 17: 287-296), All three ICG-conjugated micelles had long blood circulation with half-lives of more than 10 h, while the pHt of nanoparticles have significant effect on their blood AUC0-24 h.Moreover, our group performed an in-depth research on the effect of hydrophobicity of micellar core on its pharmacokinetics behavior, finding that lower pHt of nanoparticles would lead to longer circulation time in vivo, as shown in Figure R1.Therefore, the tumour accumulation in PGN4.9-andPGN4.3-treated groups was 1.93-and 2.06-fold of that in PGN6.3-treated group due to the higher blood AUC for PGNs with lower pHt.In other experiments from our group, the nanoparticles with lower pHt would have a smaller critical micelle concentration (Wang et al, Nature Materials 2014, 13: 204-212).Thus, the PGNs with lower pHt would have a better serum stability, which allowing for the higher tumour accumulation.
2. The authors conclude that PGN4.9 does not get activated in normal tissues, based on observations in the liver and the spleen.However, the authors should look at organs where more M2-oriented macrophages are likely to reside, such as eg the lung and possibly also the skin.
Re: We appreciate the reviewer's good suggestion and have carried out new experiments about the accumulation level and activation efficiency in other major organs, including lung, kidney, and skin.The results showed that the activation efficiency of PGN4.9 in different organs with M2-like macrophages were still significantly lower than that in tumour tissues.Therefore, we included the accumulation level and activation efficiency in different major organs in Figure 3e and Supplementary methods.Re: Thanks for the good suggestion.The proportions of CD11b + F4/80 + CD206 -macrophages were also measured by flow cytometry and the correlation analysis between M1-like macrophages content and ratiometric signal was analyzed by Graphpad Prism 8 software, as shown in Figure R2.The curve of ratiometric signal (Cy5/Cy7.5)versus the percentage of intratumoural M1-like macrophages exhibited a poor linear correlation (R = 0.2621, P = 0.2642).Combined with data in Figure 3g, the results further demonstrated the selectivity of PGN4.9 towards M2-like macrophages in vivo.Moreover, we also have verified that more than 80% of intratumoural PGN4.9 were internalized by M2-like macrophages, as shown in Figure 3f.

Fig 4b also
shows that PGN4.9 adjuvant (I presume that this is the PGN4.9 particle conjugated to IMDQ, although I don't find this very clear from the text) stimulates TLR a lot less than free IMDQ.Please explain.

Re:
We appreciate the insightful questions by the reviewer.The PGN4.9 adjuvant has been replaced with PGN4.9 nanoadjuvant we have made an explanation in the main text.For the efficacy of TLR activation, the half maximal effective concentration (EC50) of PGN4.9-IMDQ was 3.84 μM, which was significantly higher than that of free IMDQ (EC50 = 0.0124 μM).This big difference is probably due to several reasons.Firstly, the free IMDQ can diffuse freely into cells while PGN4.9 nanoadjuvant is internalized via the energy-dependent endocytosis, which is not as efficient as the former.Secondly, the internalized PGN4.9 nanoadjuvant needs to be cleaved by Cathepsin B for sustained drug release to take effect.Thirdly, all the EC50 data were obtained by static cell culture that maintain the free IMDQ level across the experiments.However, the pharmacokinetics and biodistribution of PGN4.9 nanoadjuvant is totally different from the free IMDQ in vivo.PGN4.9 nanoadjuvant can efficiently accumulate into tumour tissues, sustainably release IMDQ to selectively activate the M2-like TAMs rather than M0-and M1-like tissue-resident macrophages.In contrast, free IMDQ will distribute broadly into all the tissues after intravenous injection, and nonspecifically activate M2-like TAMs with low efficiency due to its poor tumour exposure, followed by significant off-target effect.4 should be clearer represented on the figures or in the legend.If the authors use "PGN4.9",what does that mean?I presume they mean M2 + PGN4.9, but that's nowhere stated.Could also be M0 + PGN4.9.

The experimental conditions used in Figure
Re: Thanks for the reviewer's good suggestion.To avoid misleading, we have revised the legend in Figure 4.In the reprogramming experiments, we incubated different groups of immune adjuvants with M2-like BMDMs for 24 hours, while M1-and M2-like BMDMs were the untreated cells and chosen as control groups.

Re:
Thanks for the reviewer's good suggestion.We have revised all the legend and made the experimental condition clearer.In Figure 4f, M2 group means M2-like BMDMs treated with PGN4.9 nanoreporter, whereas M2+PGN4.9 group means M2-like BMDMs was firstly treated with PGN4.9 nanoadjuvant for repolarization, followed by the treatment with PGN4.9 nanoreporter for pH-gated reporting of lysosomal acidity.

Fig 4m.
Are the MHC-I/SIINFEKL complexes higher in the PGN4.9 condition because more SIINFEKL peptide is produced or because there is a higher overall surface expression of MHC-I molecules?The authors should discriminate between these possibilities by assessing the production of SIINFEKL in M2 versus M2+PGN4.9

Fig 3 ,
Fig 3, Please also show lysosomal uptake in vivo.

6K)
Why the sudden switch from 4T1 to MC38 in this figure?All previous experiments were only demonstrated for 4T1.Did they also work with MC38? 6L/M/N) The figures show a combination therapy using anti-PD1 and PGN4.9 IMDQ.However, only tumor growth kinetics and survival are shown.Please also show infiltration data to indicate how the combination therapy might work.
6) Along the same line, What are the conditions in Figure 4f?I guess "M2" means a treatment with PGN4.9+OFF/ON fluorescent module, while "PGN4.9"means PGN4.9+IMDQ?I can only guess... 7) Fig 4m.Are the MHC-I/SIINFEKL complexes higher in the PGN4.9 condition because more SIINFEKL peptide is produced or because there is a higher overall surface expression of MHC-I molecules?The authors should discriminate between these possibilities by assessing the production of SIINFEKL in M2 versus M2+PGN4.9 8) The authors investigated the biodistribution of the particles in the tumor, but not the tumor-draining lymph node.In Fig 5, however, they suddenly look at the tdLN macrophages instead of the TAMs.What happens to the TAMs in terms of antigen processing and presentation?9) Fig 5h.Why does NPGN result in a higher killing capacity than IMDQ?10) Fig 6h.This means that these TAMs contribute to anti-tumor immunity.Do these TAMs kill cancer cells?In addition, to what extent are CD8+ T cells important?This can be shown by in vivo depletion of these cells.11) Fig 6k.The control group says "PBS", while these are simply control C57BL/6 mice.
8. The design, grouping and experimental cycle of animal experiments are not well described.How many animals in each group need to be maintained for 150 days?During this period, in addition to the change and control of tumor volume, how can animal welfare be guaranteed?Is there a humanitarian end?Are other indicators of animals tracked and monitored?Are there any abnormalities in behavior, physiological and biochemical indicators? 9.A thoroughly check should be made to improve the English and correct the grammar and typo.Reviewer #4 (Remarks to the Author): with expertise in nanomedicine, cancer immunology This manuscript by Tang et al, presents an innovative approach consisting in the use of pHgated nanoparticles with ability to deliver a drug (imiquimod) and regulate lysosomal function in tumor associated macrophages.The hypothesis, performance of experiments and results are appropriated, followed by a good presentation (both writing and figures).As the key element of this research, the development of pH-gated nanoparticles and their performance is very interesting and providing very satisfactory results, thus I recommend this manuscript for publication with minor delay after addressing the following minor concerns: 1 -Supp fig 6 legend is wrong.b) and C) have to be changed.2 -supp fig 13b) requires more details for the performance of the readout.13c) legend requires description of organs collected.It is not clear if the 4T1 model is s.c. or orthotopic (probably s.c.).These details should be clearly mentioned in the figure legend.3 -Dramatic decrease of signal is too exagerated for figure 3.h and j. or supp.fig.17.Some signal is still visible in tumors after depleting macrophages.I miss the analysis of macrophage depletion?which percentage of macrophages was depleted?only M2 or also M1 were depleted?For how long? 4 -In supp.fig.21 e-h, injected dose of IMDQ and others is missing.5 -In supp.fig.27, indication of experimental doses and times would be appreciated.6 -In supp.fig.33, dose of each treatment would be appreciated.7 -In fig.6, doses of treatments would be appreciated.8 -In supp.fig.36, time of evaluation must be indicated.

Supplementary Fig. 17 .
Accumulation level and activation efficiency of (a) PGN 6.3 and (b) PGN 4.3 in dissected livers, spleens and tumours (n = 4).Activation efficiency in different groups was obtained by normalizing to that of PGN 4.3 in tumour tissues.

Figure R3 .
Figure R3.Lysosomal pH measurement of RAW-Blue.(a) Ratiometric images of different cell lines in buffer solutions with different pH and the corresponding cells in culture medium.(b) The fitting standard curve of lysosomal pH value in RAW-Blue.(c) The lysosomal pH value of RAW-Blue with or without cytokine IL-4 (n = 16-18).

Supplementary Fig. 37 .
Quantification of tumour infiltration for different immune cells.Cell numbers of (a) CD8 + cytotoxic T cells; (b) iNOS + M1-like macrophages; (c) CD206 + M2-like macrophages; (d) CD4 + Foxp3 + regulatory T cells (n = 20-25).Statistical significance was analyzed by one-way ANOVA followed by Dunnett's multiple comparisons test 15. 6L/M/N) The figures show a combination therapy using anti-PD1 and PGN4.9 IMDQ.However, only tumor growth kinetics and survival are shown.Please also show infiltration data to indicate how the combination therapy might work.Re: We appreciate the reviewer's suggestion and have performed immune infiltration experiments in Supplementary Fig. 44.(d) Representative flow cytometry plots and (e) quantification of CD3 + CD8 + cytotoxic T lymphocytes in tumour tissues.(f, g) The percentage of CD3 + CD4 + T lymphocytes from 4T1 tumour-bearing mice with different treatments (n = 4).

3.
Fig 3g shows a correlation between the ratiometric signal and the presence of CD206 + TAMs.However, tumors may also contain M1-like TAMs.How is the correlation with those TAMs?The authors should more directly demonstrate that PGN4.9 fluoresces only within the more M2-oriented TAMs